PROJECT SUMMARY Since discovery in 2001, flagellar Ca2+ channel CatSper has remained the only Ca2+ channel in which genetic mutations cause male infertility. There are critical knowledge gaps in the current understanding of the functional regulation of this critically important channel and its signaling pathways to trigger hyperactivation, an asymmetric flagellar motion required for male fertility. The long-term goal is to deepen the understanding of sperm physiology and to uncover new pathways that could be exploited to assess and control sperm motility and fertility. CatSper channels form unique multi-protein Ca2+ signaling complexes in four linear ?racing stripe? nanodomains along the sperm tail. The presence of this spatially ordered nanodomains serves as a marker of successful sperm hyperactivation. The overall objective of this application is to elucidate how the CatSper channel complex is molecularly defined and structurally organized to integrate signal transduction pathways leading to the mechan- ical transitions required for hyperactivation. The central hypothesis is that the Ca2+ signaling state within CatSper nanodomains determines sperm motility and fertility. The rationale for determining the molecular mechanisms of CatSper channel regulation is that this knowledge will likely offer a strong scientific framework whereby new pharmacological strategies to alter sperm motility, and thus male fertility, can be developed. This application proposes to characterize novel CatSper-interacting molecules implicated in Ca2+ binding and membrane traffick- ing and microscopically visualize the Ca2+ signaling state in sperm ready to fertilize. The central hypothesis will be tested by pursuing three specific aims: 1) Define the function of the novel CatSper components in regulating channel activity and sperm motility; 2) Determine the role of the novel molecules in nanodomain formation; and 3) Elucidate the domain-integrated signaling pathways in sperm that achieve fertilization. Electrophysiological recording and flagellar waveform analysis will be employed to evaluate loss-of-function phenotype effects on channel activity and sperm motility. For the second aim, a different stage of spermatogenic cells from the knock- out models will be used to determine their role in channel complex assembly and nanodomain formation. For the third aim, motility-correlation super-resolution and in situ molecular imaging methods will be used to investigate the signaling state and domain organization of individual sperm cells with proven motility and fertility. The research proposal is innovative, in the applicant?s opinion, because it focuses on new CatSper components using new animal models to test an original concept of coupling signaling domain organization to channel activity in regulating Ca2+ signaling, and incorporates new methods into the sperm biology field. The proposed research is significant because it is expected to provide new mechanistic insights into CatSper channel activity and sperm motility regulation and molecular properties of sperm that are ready to fertilize. Ultimately, such knowledge has the potential to offer new opportunities for improvements in assisted reproduction, diagnosis of male infertility, and development of new targets for contraceptives.